The overall objective is to clarify the pathway of Beta- hydroxyisobutyrate catabolism and provide insight into the regulation of metabolism of this compound. The working hypothesis is that beta-hydroxyisobutyrate is made from valine in peripheral tissues and used by the liver for gluconeogenesis. It is proposd that 3-hydroxyisobutyrate dehydrogenase (E.C. 1.1.1.31) is important in controlling interorgan catabolism of beta-hy- droxyisobutyrate. It is further proposed that an enzyme catalyzing the same reaction as methylmalonate semialdehyde dehydrogenase (E.C. 1.2.1.27) of pseudomonads is present in mammalian tissues. Studies will be conductied with purified enzymes, isolated hepatocytes, and intact animals in different nutritional, hormonal, and developmental states. The detailed specific aims are: (a) to clone and sequence cDNA for 3-hy- droxyisobutyrate dehydrogenase; (b) to determine the effects of nutritional, hormonal, and developmental states on tissue amounts of 3-hydroxyisobutyrate dehydrogenase and its mRNA; (c) to develop an enzymatic assay for beta-hydroxyisobutyrate based on the reaction catalyzed by 3-hydroxyisobutyrate dehydrogenase; (d) to determine effects of nutritional and hormonal states on blood levels of beta-hydroxyisobutyrate; (e) to determine effects of other substrates (ethanol, oleate, beta-hydroxybutyrate) and hormones on the metabolism of beta-hydroxyisobutyrate by isolated hepatocytes; (f) to determine whether tissue specific isozymes of 3-hydroxyisobutyrate dehydrogenase exist; (g) to determine whether some enzyme other than 3-hydroxyisobutyrate dehydrogenase is responsible for the oxidation of R-3- hydroxyisobutyrate (intermediate in thymine catabolism); (h) to identify cysteine residue(s) involved in substrate binding by 3- hydroxyisobutyrate dehydrogenase; (i) to isolate and characterize rat liver methylmalonate semialdehyde dehydrogenase; (j) to clone and sequence cDNA for methylmalonate semialdehyde dehydrogenase; and (k) to determine the effects of nutritional, hormonal, and developmental states on tissue amounts of methylmalonate semialdehyde dehydrogenase and its mRNA. These studies should further our understanding of the possible role of beta-hydroxyisobutyrate as a source of carbon for hepatic gluconeogenesis, establish whether methylmalonate semialdehyde dehydrogenase is present in tissues of higher organisms, and explain the accumulation of beta-hydroxyisobutyrate and related compounds under conditions of ketoacidosis and in various inherited organic acidemias.